Although in genetic engineering techniques numerous polypeptide expression systems for prokaryotic and eukaryotic hosts are already known, there is a continuous need for novel systems which may have advantages over the known systems.
Very widely used are the prokaryotic Escherichia coli host and the eukaryotic yeast host, e.g. Saccharomyces cerevisiae, for which a high number of different expression hybrid vectors, mostly plasmids, have been developed. The drawbacks of E. coli hosts are that they cannot glycosylate the formed polypeptide and that for lack of secretion the foreign peptide may accumulate within the host cell and prevent further growth. The yeast hosts do glycosylate, however, like E. coli, they do not secrete the polypeptides, except very small ones, into the nutrient medium. Yeasts secrete only into the periplasmic space. Higher eukaryotic hosts are mammalian cancer cells which are able to glycosylate and secrete into the nutrient medium, however, cultivation thereof is very slow and expensive and the danger exists that oncogenic nucleic acids are isolated together with the desired peptide of which the latter may not be freed.
In the need for other hosts also filamentous fungi, such as Neurospora crassa, Aspergillus nidulans and Aspergillus niger, have been investigated. Such fungi are already widely used for industrial purposes, however, the application thereof in genetic engineering techniques has lagged behind, mainly for lack of an appropriate transformation system. In contrast to Saccharomyces cerevisiae, filamentous fungi do not contain plasmids which could be used for the introduction of foreign genes and phenotype selection. It is, however, possible to transform filamentous fungi with foreign plasmids containing a selectable marker gene. All vectors described so far for filamentous fungi do not autonomously replicate, as those of yeasts do, but are integrated into the fungal chromosome. This event occurs only at a very low frequency. Advantageously, on the other hand, integrative transformation renders the transformants mitotically very stable, even under non-selective conditions. Stable integration of more than one hundred copies has been reported.
The first vector for filamentous fungi described contained the qa-2 gene of Neurospora crassa as selectable marker. This gene encodes the enzyme catabolic dehydroquinase and can be used for functional complementation of aro mutants of N. crassa [Case, M. E., Schweizer, M., Kushner, S. R. and Giles, N. H. (1979) Proc. Natl. Acad. Sci. USA 76, 5259-5263]. Aro mutants are unable to grow on minimal medium without an aromatic amino acid supplement. Transformation of N. crassa by the qa-2 vector occurred by integration of a single copy of the plasmid into the chromosome. 30% of stable Aro.sup.+ integrants retained the integrated qa-2 gene still linked to the bacterial plasmid sequences [Case, M. E. (1982) in Genetic Engineering of Microorganisms for Chemicals (Hollander, A., DeMoss, D., Kaplan, S., Konisky, J., Savage, D. and Wolfe, R. S., eds), pp. 87-100, Plenum]. This observation made cotransformation of non-selective DNA-sequences together with selective ones a feasible task.
In Aspergillus nidulans, which has a sexual cycle and is therefore amenable to classical genetic manipulations, both negative and positive selection systems have been identified. Either using heterologous DNA from N. crassa or homologous DNA, functional complementation of A. nidulans pyrG-mutants by transformation with plasmids containing the pyrG gene was obtained (Ballance et al. BBRC 112,284 1983; Tilburn et al. Gene 26, 205, 1983). In other systems mutations at the trpC or argB locus were functionally complemented by transformation with the appropriate plasmids [Yelton et al. PNAS 81, 1470, 1984; Yelton et Timberlake J. Cell. Biochem. Suppl. 9C 173, 1985; Johnstone et al. EMBO J. 4, 1307, 1983].
A dominant positive selection system has also been developed making use of the amdS gene isolated from A. nidulans which enables A. niger transformed therewith to grow on acetamide as sole nitrogen source (Tilburn et al., Gene 26, 205, 1983; Wernars et al., Curr. Genet. 9, 361, 1985; Kelly, J. M. et al., EMBO J. 4, 475,1985).
Compared to N. crassa or A. nidulans, A. niger is by far the more important organism. It is used widely in the industrial production of enzymes, e.g. for use in the food industry. A. niger differs from A. nidulans by its secretory capacity, in that it secretes a variety of hydrolytic enzymes, e.g. glucoamylase, .alpha.-amylase, pectinase, cellulase, .beta.-glucanase, .beta.-galactosidase, naringinase, pentosanase, acid protease and lignase, the glucoamylase and pectinase complex being the most important ones.
A. niger has no known sexual cycle. Mutations can therefore not be introduced via meiotic recombinations. By classical mutation and selection procedures, extensive strain improvements in the secretion of hydrolytic enzymes have however been achieved.
Of the genes of A. niger enzymes only those of glucoamylase (Boel et al. EMBO J. 3, 1581, 1984) and alcohol and aldehyde dehydrogenase (WO 86/06097) together with their promoter and signal sequences have been characterised and used in transformation experiments with A. nidulans and A. niger, respectively.
As selection markers for A. niger have been used the heterologous amds gene (Kelly and Hynes, EMBO 3. 4, 475, 1985), and the argB gene (Buxton et al., Gene 37, 207, 1985; EP 184 438; WO 86/06097), both obtained from A. nidulans.
A. niger is the most important organism for the industrial production of pectin degrading enzymes. Pectins are polygalacturonides of high molecular weight (20000-40000 D) consisting of .alpha.-1,4-glycosidic bounded D-galacturonic acid polymers and occur in nature as constituents of higher plant cells, where they are attached to cellulose molecules and where they are mainly found in the primary cell wall and the middle lamella. Amongst the richest sources of pectin are lemon and orange rind, which contain about 30% of this polysaccharide. Pectic enzymes are degrading the carbohydrate polymer substrate either by hydrolysis of the .alpha.-1,4-glycosidic bond (polygalacturonase) or by transelemination of the .alpha.-4,5 unsaturated galacturonic residue from the pectin molecule (different pectin lyases). The systematic name of pectin lyase is pectin transeliminase (EC 4.2.2.10).
In A. niger the proteins of the pectic complex are not expressed constitutively. Under inducing conditions using pectin or breakdown products thereof A. niger expresses the above mentioned enzymes, including PLI, when other carbon sources, such as glucose or sucrose, are limiting. In surface cultures the pectic enzymes tend to remain associated with the outer cell wall. Increasing pectin and Ca.sup.2+ concentration in the medium leads to complete secretion.
Pectinases, such as PLI, are used by the food stuff industry mainly for fruit juice clarification.
From A. niger two different pectin lyases, PLI and PLII, have been purified and partially characterized by F. E. A. Van Houdenhoven (22). PLI contains four residues of mannose, whereas PLII has two residues of mannose and glucose each. The enzymes have different molecular weights (PLI: 37.5 kD, PLII: 36 kD). Before the present invention no total or partial amino acid sequences have been published.
The present invention is based on a partial structure determination of pectin lyase I (PLI) which allowed the synthesis of DNA probes coding for relevant parts of the protein. By means of the DNA probes it was possible to screen for and isolate DNA coding for PLI, eventually together with pre- and post-sequences thereof, from a gene library of A. niger.
By hybridization of parts of the PLI gene to a genomic library of A. niger further PL genes have been detected which are also subject of the present invention. The PLI structural gene with the N-terminus flanking region obtained from A. niger N756 is at its N-terminus identical to the PLD gene obtained from A. niger N400. Accordingly the latter is also part of the present invention. The present PLA seems to be part of the previously purified PL mixture named PLII.
Hereinafter, PLI is also named PLD and the PLI structural gene is named pelD.